Conformational Dynamics in Glutathione S-Transferase

谷胱甘肽 S-转移酶的构象动力学

基本信息

批准号：
6436574
负责人：
WILLIAM M ATKINS
金额：
$ 23.8万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-01-10 至 2005-12-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The glutathione S-transferases (GSTs) are a family of detoxification enzymes that metabolize environmental xenobiotics and drugs, including anti-cancer agents, by conjugating them to the tripeptide glutathione (GSH). GSTs also modulate oxidative stress by metabolizing lipid hydroperoxides and lipid hydroxy-enals. GSTs are likely to play a role in sensitivity to atherosclerosis, cataracts, and neurodegenerative diseases. As a canonical family of structurally related proteins, the GSTs provide a model for understanding the evolution of substrate diversity, which apparently correlates with the evolution of protein dynamics in some GSTs. The GSTA1-1 isoform has two unusual features that may uniquely contribute to its catalytic diversity as a detoxification enzyme. One feature is a catalytic Tyr with an unusually low pKa, which, possibly, provides electrostatic forces and increases solvation of the active site. The ionization state of this Tyr does not change during chemical steps of the catalytic cycle, and the function of the unusual ionization properties remains unknown. The second feature is a dynamic C-terminal helix, which undergoes ligand-dependent redistribution between 'open' and 'closed' conformations. Highly related, nearly structurally identical, GSTs possess C-terminal helices that are 'static' and remain either 'open' or 'closed.' This proposal explores the catalytic function of the Tyr ionization properties and of the C-terminal helix and, in particular, the hypothesis that the two features have co-evolved as an evolutionary bridge between primitive GSTs and highly evolved substrate specific isoforms. In order to understand the structure, function, and dynamics of the GST family, the specific aims of this proposal are: 1) to determine the stage of GSTA1-1 catalysis at which the C-terminus closes; 2) to determine the function of the unusual ionization properties of the active site Tyr; 3) to explore the molecular determinants of substrate diversity by directed evolution of GSTA1-1 and directed de-evolution of GSTA4-4. The techniques to be used include x-ray crystallography, NMR, and fluorescence of model ternary complexes, to monitor the C-terminal structure and dynamics. In order to determine whether the C-terminus must be closed in the transition state for the chemical step, linear free energy relationships will be exploited. The relationship, if any, between C-terminal dynamics and the ionization of the catalytic Tyr will be explored with stopped-flow kinetic approaches and steady state fluorescence with an engineered Trp reporter.

描述（由申请人提供）：谷胱甘肽 S-转移酶 (GST) 是代谢环境异生物质的解毒酶家族和药物，包括抗癌剂，通过将它们与三肽缀合谷胱甘肽（GSH）。 GST 还通过代谢脂质来调节氧化应激氢过氧化物和脂质羟基烯醛。商品及服务税 (GST) 可能会在以下方面发挥作用：对动脉粥样硬化、白内障和神经退行性疾病的敏感性。作为一个作为结构相关蛋白的典型家族，GST 提供了一个模型了解底物多样性的演变，这显然与随着一些 GST 中蛋白质动力学的演变。 GSTA1-1 同工型具有两个不寻常的特征可能对其催化多样性有独特贡献：一种解毒酶。一个特点是催化 Tyr 具有异常低的 pKa，可能提供静电力并增加溶剂化活性位点。该 Tyr 的电离态在过程中不会改变催化循环的化学步骤，以及不寻常的功能电离特性仍然未知。第二个特点是动态 C 末端螺旋，在之间进行配体依赖性重新分配 “开放”和“封闭”构象。高度相关，几乎在结构上相同，GST 具有“静态”的 C 端螺旋，并且保持 “开放”或“封闭”。该提案探讨了 Tyr 的催化功能电离特性和 C 末端螺旋，特别是假设这两个特征作为进化桥梁共同进化原始 GST 和高度进化的底物特异性亚型之间。为了为了了解 GST 系列的结构、功能和动态，该提案的具体目标是： 1) 确定 GSTA1-1 的阶段 C 末端关闭的催化作用； 2) 确定函数活性位点 Tyr 不寻常的电离特性； 3）探索 GSTA1-1 定向进化决定底物多样性的分子决定因素以及 GSTA4-4 的定向去进化。所使用的技术包括 X 射线模型三元配合物的晶体学、核磁共振和荧光，以监测 C端结构和动力学。为了确定是否 C 末端必须在化学步骤的过渡态下闭合，线性自由能源关系将被利用。之间的关系（如果有的话）将探索催化 Tyr 的 C 末端动力学和电离采用停流动力学方法和稳态荧光设计的色氨酸报告基因。